Thirty-three melanocyte cell strains established from newborn skin (foreskins) were grown over 1 year in culture to determine the lifespan of cultured melanocytes. In addition, 20 melanocyte cell cultures were established from adult facial skin and from breast skin. They could be carried in vitro for only four to five passages, corresponding to approximately eight population doublings. It was found that potent melanocyte growth stimulators are substances such as 12-myristate acetate (PMA), mezerin, teleocidin, aplysiatoxin, and lyngbyatoxin. Different factors and hormones were tested for their growth promoting activity for normal melanocytes. These include epidermal growth factor (EGF), nerve growth factor (NGF), fibroblast growth factor (FGF), melanocyte stimulating hormone (MSH), pig brain extract, and dermal extract. Only pig dermal extract mildly stimulated melanocyte growth and sustained melanocytes for up to 6 months in culture. DNA content of three melanocyte strains grown in the presence of PMA at passages 10, 19, and 27 was studied by cytofluorometry. It was found that in all instances melanocytes had diploid (2C) DNA content. Cytogenetic analysis performed by the trypsin-Giemsa banding technique on two melanocyte cell strains both at passage 18 showed a diploid and near diploid normal karyotype. To further define the phenotype of melanocytes, newborn and adult melanocytes were tested with a panel of monoclonal antibodies against 37 cell-surface antigens of malignant melanoma. Four categories of antigens were distinguished on the basis of their expression on melanocytes. Eight anti-melanocyte monoclonal antibodies were developed and shown to react with normal melanocytes. Four of these are broadly reactive; four are restricted and react with melanocytes and some melanomas. One is reactive specifically with adult melanocytes only. (M)